The present invention relates to chimeric receptors containing one or more regions homologous to a metabotropic glutamate receptor and one or more regions homologous to a calcium receptor.
The following description provides a summary of information relevant to the present invention. It is not an admission that any of the information provided herein is prior art to the presently claimed invention, nor that any of the publications specifically or implicitly referenced are prior art to that invention.
Glutamate is the major excitatory neurotransmitter in the mammalian brain. Glutamate produces its effects on central neurons by binding to and thereby activating cell surface receptors. These receptors have been subdivided into two major classes, the ionotropic and metabotropic glutamate receptors, based on the structural features of the receptor proteins, the means by which the receptors transduce signals into the cell, and pharmacological profiles.
The ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that, upon binding glutamate, open to allow the selective influx of certain monovalent and divalent cations, thereby depolarizing the cell membrane. In addition, certain iGluRs with relatively high calcium permeability can activate a variety of calcium-dependent intracellular processes. These receptors are multisubunit protein complexes that may be homomeric or heteromeric in nature. The various iGluR subunits all share common structural motifs, including a relatively large amino-terminal extracellular domain (ECD), followed by a multiple transmembrane domain (TMD) comprising two membrane-spanning regions (TMs), a second smaller intracellular loop, and a third TM, before terminating with an intracellular carboxy-terminal domain (CT). Historically the iGluRs were first subdivided pharmacologically into three classes based on preferential activation by the agonists alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA), kainate (KA), and N-methyl-D-aspartate (NMDA). Later, molecular cloning studies coupled with additional pharmacological studies revealed a greater diversity of iGluRs, in that multiple subtypes of AMPA, KA and NMDA receptors are expressed in the mammalian CNS (Hollman and Heinemann, Ann. Rev. Neurosci. 7:31, 1994).
The metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors capable of activating a variety of intracellular second messenger systems following the binding of glutamate or other potent agonists including quisqualate and 1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) (Schoepp et al., Trends Pharmacol. Sci. 11:508, 1990; Schoepp and Conn, Trends Pharmacol. Sci. 14:13, 1993).
Activation of different metabotropic glutamate receptor subtypes in situ elicits one or more of the following responses: activation of phospholipase C, increases in phosphoinositide (PI) hydrolysis, intracellular calcium release, activation of phospholipase D, activation or inhibition of adenylyl cyclase, increases and decreases in the formation of cyclic adenosine monophosphate (cAMP), activation of guanylyl cyclase, increases in the formation of cyclic guanosine monophosphate (cGMP), activation of phospholipase A2, increases in arachidonic acid release, and increases or decreases in the activity of voltage- and ligand-gated ion channels (Schoepp and Conn, Trends Pharmacol. Sci. 14:13, 1993; Schoepp, Neurochem. Int. 24:439, 1994; Pin and Duvoisin, Neuropharmacology 34:1, 1995).
Thus far, eight distinct mGluR subtypes have been isolated via molecular cloning, and named mGluR1 to mGluR8 according to the order in which they were discovered (Nakanishi, Neuron 13:1031, 1994, Pin and Duvoisin, Neuropharmacology 34:1, 1995; Knopfel et al., J. Med. Chem. 38:1417, 1995). Further diversity occurs through the expression of alternatively spliced forms of certain mGluR subtypes (Pin et al., PNAS 89:10331, 1992; Minakami et al., BBRC 199:1136, 1994). All of the mGluRs are structurally similar, in that they are single subunit membrane proteins possessing a large amino-terminal extracellular domain (ECD) followed by seven putative transmembrane domain (7TMD) comprising seven putative membrane spanning helices connected by three intracellular and three extracellular loops, and an intracellular carboxy-terminal domain of variable length (cytoplasmic tail) (CT) (see, Schematic FIG. 1a).
The eight mGluRs have been subdivided into three groups based on amino acid sequence identities, the second messenger systems they utilize, and pharmacological characteristics (Nakanishi, Neuron 13:1031, 1994; Pine and Duvoisin, Neuropharmacology 34:1, 1995; Knopfel et al., J. Med. Chem. 38:1417, 1995). The amino acid identity between mGluRs within a given group is approximately 70% but drops to about 40% between mGluRs in different groups. For mGluRs in the same group, this relatedness is roughly paralleled by similarities in signal transduction mechanisms and pharmacological characteristics.
The Group I mGluRs comprise mGluR1, mGluR5and their alternatively spliced variants. The binding of agonists to these receptors results in the activation of phospholipase C and the subsequent mobilization of intracellular calcium. For example, Xenopus oocytes expressing recombinant mGluR1 receptors have been utilized to demonstrate this effect indirectly by electrophysiological means (Masu et al., Nature 349:760, 1991; Pin et al., PNAS 89:10331, 1992). Similar results were achieved with oocytes expressing recombinant mGluR5 receptors (Abe et al., J. Biol. Chem. 267:13361, 1992; Minakami et al., BBRC 199:1136, 1994). Alternatively, agonist activation of recombinant mGluR1 receptors expressed in Chinese hamster ovary (CHO) cells stimulated PI hydrolysis, cAMP formation, and arachidonic acid release as measured by standard biochemical assays (Aramori and Nakanishi, Neuron 8:757, 1992). In comparison, activation of mGluR5 receptors expressed in CHO cells stimulated PI hydrolysis and subsequent intracellular calcium transients but no stimulation of cAMP formation or arachidonic acid release was observed (Abe et al., J. Biol. Chem. 267:13361, 1992). The agonist potency profile for Group I mGluRs is quisqualate greater than glutamate=ibotenate greater than (2S,1xe2x80x2S,2xe2x80x2S)-2-carboxycyclopropyl)glycine (L-CCG-I) greater than (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD). Quisqualate is relatively selective for Group I receptors, as compared to Group II and Group III mGluRs, but it also potently activates ionotropic AMPA receptors (Pin and Duvoisin, Neuropharmacology, 34:1, Knopfel et al., J. Med. Chem. 38:1417, 1995).
The Group II mGluRs include mGluR2 and mGluR3. Activation of these receptors as expressed in CHO cells inhibits adenylyl cyclase activity via the inhibitory G protein, Gi, in a pertussis toxin-sensitive fashion (Tanabe et al., Neuron 8:169, 1992; Tanabe et al., Neurosci. 13:1372, 1993). The agonist potency profile for Group II receptors is L-CCG-I greater than glutamate greater than ACPD greater than ibotenate greater than quisqualate. Preliminary studies suggest that L-CCG-I and (2S,1xe2x80x2R,2xe2x80x2R,3xe2x80x2R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV) are both relatively selective agonists for the Group II receptors (Knopfel et al., J. Med. Chem. 38:1417, 1995).
The Group III mGluRs include mGluR4, mGluR6, mGluR7 and mGluR8. Like the Group II receptors these mGluRs are negatively coupled to adenylate cyclase to inhibit intracellular cAMP accumulation in a pertussis toxin-sensitive fashion when expressed in CHO cells (Tanabe et al., J. Neurosci. 13:1372, 1993; Nakajima et al., J. Biol. Chem. 268:11868, 1993; Okamoto et al., J. Biol. Chem. 269:1231, 1994; Duvoisin et al., J. Neurosci. 15:3075, 1995). As a group, their agonist potency profile is (S)-2-amino-4-phosphonobutyric acid (L-AP4) greater than glutamate greater than ACPD greater than quisqualate, but mGluR8 may differ slightly with glutamate being more potent than L-AP4 (Knopfel et al., J. Med. Chem. 38:1417, 1995; Duvoisin et al., J. Neurosci. 15:3075, 1995). Both L-AP4 and (S)-serine-O-phosphate (L-SOP) are relatively selective agonists for the Group III receptors.
Finally, the eight mGluR subtypes have unique patterns of expression within the mammalian CNS that in many instances are overlapping (Masu et al., Nature 349:760, 1991; Martin et al., Neuron 9:259, 1992; Ohishi et al., Neurosci. 53:1009, 1993; Tanabe et al., J. Neurosci. 13:1372; Ohishi et al., Neuron 13:55, 1994, Abe et al., J. Biol. Chem. 267:13361, 1992; Nakajima et al., J. Biol. Chem. 268:11868, 1993; Okamoto et al., J. Biol. Chem. 269:1231, 1994; Duvoisin et al., J. Neurosci. 15:3075, 1995). As a result certain neurons may express only one particular mGluR subtype, while other neurons may express multiple subtypes that may be localized to similar and/or different locations on the cell (i.e., postsynaptic dendrites and/or cell bodies versus presynaptic axon terminals). Therefore, the functional consequences of mGluR activation on a given neuron will depend on the particular mGluRs being expressed; the receptors"" affinities for glutamate and the concentrations of glutamate the cell is exposed to; the signal transduction pathways activated by the receptors; and the locations of the receptors on the cell. A further level of complexity may be introduced by multiple interactions between mGluR expressing neurons in a given brain region. As a result of these complexities, and the lack of subtype-specific mGluR agonists and antagonists, the roles of particular mGluRs in physiological and pathophysiological processes affecting neuronal function are not well defined. Still, work with the available agonists and antagonists have yielded some general insights about the Group I mGluRs as compared to the Group II and Group III mGluRs.
Attempts at elucidating the physiological roles of Group I mGluRs suggest that activation of these receptors elicits neuronal excitation. Various studies have demonstrated that ACPD can produce postsynaptic excitation upon application to neurons in the hippocampus, cerebral cortex, cerebellum, and thalamus as well as other brain regions. Evidence indicates that this excitation is due to direct activation of postsynaptic mGluRs, but it has also been suggested to be mediated by activation of presynaptic mGluRs resulting in increased neurotransmitter release (Baskys, Trends Pharmacol. Sci. 15:92, 1992; Schoepp, Neurochem. Int. 24:439, 1994; Pin and Duvoisin, Neuropharmacology 34:1). Pharmacological experiments implicate Group I mGluRs as the mediators of this excitation. The effect of ACPD can be reproduced by low concentrations of quisqualate in the presence of iGluR antagonists (Hu and Storm, Brain Res. 568:339, 1991; Greene et al. Eur. J. Pharmacol. 226:279, 1992), and two phenylglycine compounds known to activate mGluR1, (S)-3-hydroxyphenylglycine ((S)-3HPG) and (S)-3,5-dihydroxyphenylglycine ((S)-DHPG), also produce the excitation (Watkins and Collingridge, Trends Pharmacol. Sci. 15:333, 1994). In addition, the excitation can be blocked by (S)-4-carboxyphenylglycine ((S)-4CPG), (S)-4-carboxy-3-hydroxyphenylglycine ((S)-4C3HPG) and (+)-alpha-methyl-4-carboxyphenylglycine ((+)-MCPG), compounds known to be mGluR1 antagonists (Eaton et al., Eur. J. Pharmacol. 244:195, 1993; Watkins and Collingridge, Trends Pharmacol. Sci. 15:333, 1994).
Other studies examining the physiological roles of mGluRs indicate that activation of presynaptic mGluRs can block both excitatory and inhibitory synaptic transmission by inhibiting neurotransmitter release (Pin and Duvoisin, Neuropharmacology 34:1). Presynaptic blockade of excitatory synaptic transmission by ACPD has been observed on neurons in the visual cortex, cerebellum, hippocampus, striatum and amygdala (Pin et al., Curr. Drugs: Neurodegenerative Disorders 1:111, 1993), while similar blockade of inhibitory synaptic transmission has been demonstrated in the striatum and olfactory bulb (Calabresi et al., Neurosci. Lett. 139:41, 1992; Hayashi et al., Nature 366:687, 1993). Multiple pieces of evidence suggest that Group II mGluRs mediate this presynaptic inhibition. Group II mGluRs are strongly coupled to inhibition of adenylyl cyclase, like alpha2-adrenergic and SHT1A-serotonergic receptors which are known to mediate presynaptic inhibition of neurotransmitter release in other neurons. The inhibitory effects of ACPD can also be mimicked by L-CCG-I and DCG-IV, which are selective agonists at Group II mGluRs (Hayashi et al., Nature 366:687, 1993; Jane et al., Br. J. Pharmacol. 112:809, 1994). Moreover, it has been demonstrated that activation of mGluR2 can strongly inhibit presynaptic, N-type calcium channel activity when the receptor is expressed in sympathetic neurons (Ikeda et al., Neuron 14:1029, 1995), and inactivation of these channels is known to inhibit neurotransmitter release. Finally, it has been observed that L-CCG-I, at concentrations selective for Group II mGluRs, inhibits the depolarization-evoked release of 3H-aspartate from rat striatal slices (Lombardi et al., Br. J. Pharmacol. 110:1407, 1993). Evidence for physiological effects of Group II mGluR activation at the postsynaptic level is limited. However, one study suggests that postsynaptic actions of L-CCG-I can inhibit NMDA receptor activation in cultured mesencephalic neurons (Ambrosini et al., Mol. Pharmacol. 47:1057, 1995).
Physiological studies have demonstrated that L-AP4 can also inhibit excitatory synaptic transmission on a variety of CNS neurons. Included are neurons in the cortex, hippocampus, amygdala, olfactory bulb and spinal cord (Koerner and Johnson, Excitatory Amino Acid Receptors; Design of Agonists and Antagonists p. 308, 1992; Pin et al., Curr. Drugs: Neurodegenerative Disorders 1:111, 1993). The accumulated evidence indicates that the inhibition is mediated by activation of presynaptic mGluRs. Since the effects of L-AP4 can be mimicked by L-SOP, and these two agonists are selective for Group III mGluRs, members of this mGluR group are implicated as the mediators of the presynaptic inhibition (Schoepp, Neurochem. Int. 24:439, 1994; Pin and Duvoisin, Neuropharmacology 34:1). In olfactory bulb neurons it has been demonstrated that L-AP4 activation of mGluRs inhibits presynaptic calcium currents (Trombley and Westbrook, J. Neurosci. 12:2043, 1992). It is therefore likely that the mechanism of presynaptic inhibition produced by activation of Group III mGluRs is similar to that for Group II mGluRs, i.e., blockade of N-type calcium channels and inhibition of. neurotransmitter release. L-AP4 is also known to act postsynaptically to hyperpolarize ON bipolar cells in the retina. It has been suggested that this action may be due to activation of a mGluR, which is coupled to the cGMP phosphodiesterase in these cells (Schoepp, Neurochem. Int. 24:439, 1994; Pin and Duvoisin, Neuropharmacology 34:1).
Metabotropic glutamate receptor activation studies using agonists, antagonists and recombinant vertebrate cell lines expressing mGluRs have been used to evaluate the cellular effects of the stimulation and the inhibition of different metabotropic glutamate receptors. For example, agonist stimulation of mGluR1 expressed in Xenopus oocytes demonstrated coupling of receptor activation to mobilization of intracellular calcium as assessed indirectly using electrophysiology techniques (Masu et al., Nature 349:760-765, 1991). Agonist stimulation of mGluR1 expressed in CHO cells stimulated PI hydrolysis, cAMP formation and arachidonic acid release (Aramori and Nakanishi, Neuron 8:757-765, 1992). Agonist stimulation of mGluR5 expressed in CHO cells also stimulated PI hydrolysis which was shown to be associated with a transient increase in cytosolic calcium as assessed by loading cells with the fluorescent calcium chelator fura-2 (Abe et al., J. Biol. Chem. 267:13361-13368, 1992). Agonist-induced activation of mGluR1 and mGluR5 induced PI hydrolysis in CHO cells was not antagonized by AP3 and AP4, which are both antagonists of glutamate-stimulated PI hydrolysis in situ (Nicoletti et al., Proc. Natl. Acad. Sci. USA 833:1931-1935, 1986; Schoepp and Johnson, J. Neurochem. 53:273-278, 1989). Agonist stimulation of CHO cells expressing mGluR2 (Tanabe et al., Neuron 8:169-179, 1992) or mGluR7 (Okamoto et al., J. Biol. Chem. 269:1231-1236, 1994) resulted in receptor-mediated inhibition of cAMP formation and also confirmed the ligand specificity previously observed in situ. Studies using agonists were also carried out in conjunction with site-directed mutagenesis to reveal specific amino acids playing important roles in glutamate binding (O""Hara et al., Neuron 11:41-52, 1993).
Metabotropic glutamate receptors (mGluRs) have been implicated in a variety of neurological pathologies including stroke, head trauma, spinal cord injury, epilepsy, ischemia, hypoglycemia, anoxia, and neurodegenerative diseases such as Alzheimer""s disease (Schoepp and Conn, Trends Pharmacol. Sci. 14:13, 1993; Cunningham et al., Life Sci. 54: 135, 1994; Pin et al., Neuropharmacology 34:1, 1995; Knopfel et al., J. Med. Chem. 38:1417, 1995;). A role for metabotropic glutamate receptors in nociception and analgesia has also been demonstrated (Meller et al., Neuroreport 4:879, 1993). Metabotropic glutamate receptors have also been shown to be required for the induction of hippocampal long-term potentiation and cerebellar long-term depression (Bashir et al., Nature 363:347, 1993; Bortolotto et al., Nature 368:740, 1994; Aiba et. al. Cell 79: 365 and Cell 79: 377, 1994).
Metabotropic glutamate receptor agonists have been reported to have effects on various physiological activities. For example, trans-ACPD was reported to possess both proconvulsant and anticonvulsant effects (Zheng and Gallagher, Neurosci. Lett. 125:147, 1991; Sacaan and Schoepp, Neurosci. Lett. 139:77, 1992; Taschenberger et al., Neuroreport 3:629, 1992; Sheardown, Neuroreport 3:916, 1992), and neuroprotective effects in vitro and in vivo (Pizzi et al., J. Neurochem. 61:683, 1993; Koh et al., Proc. Natl. Acad. Sci. USA 88:9431, 1991; Birrell et al., Neuropharmacol. 32:1351, 1993; Siliprandi et al., Eur. J. Pharmacol. 219:173, 1992; Chiamulera et al., Eur. J. Pharmacol. 216:335, 1992). The metabotropic glutamate receptor antagonist L-AP3 was shown to protect against hypoxic injury in vitro (Opitz and Reymann, Neuroreport 2:455, 1991). A subsequent study reported that trans-ACPD produced neuroprotection which was antagonized by L-AP3 (Opitz and Reymann, Neuropharmacol. 32:103, 1993). (5)-4C3HPG was shown to protect against audiogenic seizures in DBA/2 mice (Thomasen et al., J. Neurochem. 62:2492, 1994). Other modulatory effects expected of metabotropic glutamate receptor modulators include synaptic transmission, neuronal death, neuronal development, synaptic plasticity, spatial learning, olfactory memory, central control of cardiac activity, waking, control of movements, and control of vestibulo ocular reflex (for reviews, see Nakanishi, Neuron 13:1031-37, 1994; Pin et al., Neuropharmacology 34:1, 1995; Knopfel et al., J. Med. Chem. 38:1417, 1995).
The structures of mGluR-active molecules currently known in the art are limited to amino acids which appear to act by binding at the glutamate binding site (Pin, et al, Neuropharmacology 34:1, 1995; Knopfel et al., J. Med. Chem. 38:1418). This limits the range of pharmacological properties and potential therapeutic utilities of such compounds. Furthermore, the range of pharmacological specificities associated with these mGluR-active molecules does not allow for complete discrimination between different subtypes of metabotropic glutamate receptors (Pin et al., Neuropharmacology 34:1, 1995 and Knopfel et al., J. Med. Chem. 38:1418). Rapid progress in the field of mGluR-active molecules cannot be made until more potent and more selective mGluR agonists, antagonists and modulators are discovered (Pin et al., Neuropharmacology 34:1, 1995; Knopfel et al., J. Med. Chem. 38:1418). Indeed, no mGluR-active molecules are presently under clinical development. High throughput functional screening of compounds and compound libraries using cell lines expressing individual mGluRs represents an important approach to identifying such novel compounds (Knopfel et al., J. Med. Chem. 38:1418).
Several laboratories have constructed cell lines expressing metabotropic glutamate receptors which appear to function appropriately (Abe et al., J. Biol. Chem. 267:13361, 1992; Tanabe et al., Neuron 8:169, 1992; Aramori and Nakanishi, Neuron 8:757, 1992, Nakanishi, Science 258:597, 1992;.Thomsen et al., Brain Res. 619:22, 1992; Thomsen et al., Eur. J. Pharmacol. 227:361, 1992; O""Hara et al., Neuron 11:41, 1993; Nakjima et al., J. Biol. Chem. 268:11868, 1993; Tanabe et al., J. Neurosci. 13:1372, 1993; Saugstad et al., Mol. Pharmacol. 45:367, 1994; Okamoto et al., J. Biol. Chem. 269:1231, 1994; Gabellini et al., Neurochem. Int. 24:533, 1994; Lin et al., Soc. Neurosci. Abstr. 20:468, 1994; Flor et al., Soc. Neurosci. Abstr. 20:468, 1994; Flor et al., Neuropharmacology 34:149, 1994). Other reports have noted that expression of functional mGluR expressing cell lines is not predictable. For example, Tanabe et al., (Neuron 8:169, 1992) were unable to demonstrate functional expression of mGluR3 and mGluR4, and noted difficulty obtaining expression of native mGluR1 in CHO cells. Gabellini et al., (Neurochem. Int. 24:533, 1994) also noted difficulties with mGluR1 expression in HEK 293 cells and it is possible that some of these difficulties may be due to desensitization characteristics of these receptors. Furthermore, screening methodologies useful for identification of compounds active at Class I mGluRs are not readily amenable to identification of compounds active at class II and III mGluRs and vice versa due to the differences in second messenger coupling. Finally, mGluRs have been noted to rapidly desensitize upon agonist stimulation which may adversely affect the viability of cell lines expressing these receptors and makes the use of native mGluRs for screening difficult.
Different G-protein coupled receptors exhibit differential ligand affinities and coupling to second messengers. G-protein coupled receptors all have a similar structure: an N-terminal extracellular domain (ECD), a seven-transmembrane domain (7TMD) comprising seven membrane spanning helices and therefore defining three intracellular and three extracellular loops, and a cytoplasmic tail (CT), but differ in the exact sequences comprising each region. These sequence differences are thought to provide the specificity of receptor interactions with ligands of different chemical compositions and receptor interaction with different G-proteins. Construction of chimeric receptors in which small peptide segments from related receptors are exchanged using recombinant DNA techniques has proven a useful technique to assess the participation of different sequence regions in determining this specificity. For example, exchanging the third intracellular loops between various adrenergic, muscarinic acetylcholine and angiotensin receptors results in conversion of G-protein coupling specificity. Thus, receptors whose activation normally results in inhibition or activation of adenylate cyclase can be converted to receptors with the same or similar ligand binding properties but whose activation leads to stimulation of phospholipase-C and vice versa (Kobilka et al., Science 240:1310, 1988; Wess et al., FEBS Lett. 258:133, 1989; Cotecchia et al., Proc. Nat""l. Acad. Sci. U.S.A. 87:2896, 1990; Lechleiter et al., EMBO J. 9:4381, 1990; Wess et al., Mol. Pharmacol. 38:517, 1990; Wong et al., J. Biol. Chem. 265:6219, 1990; Cotecchia et al., J. Biol. Chem. 267:1633, 1992; Wang et al., J. Biol. Chem. 270:16677, 1995). In these receptors which share the third intracellular loop plays an important role in determining the specificity of G-protein coupling. While such experiments indicate that the third intracellular loop plays an important role in determining the specificity of G protein coupling in these related receptors, they have failed to identify any specific amino acid sequence motif which is responsible. In addition, the third intracellular loop has been shown to be at least partly responsible for desensitization of such receptors (Okamoto et al., Cell 67:723, 1991; Liggett et al., J. Biol. Chem. 267:4740, 1992).
Metabotropic glutamate receptors are related to other G-protein coupled receptors in overall topology, but not in specific amino acid sequence. An unusual feature of mGluRs is their very large ECDs (ca. 600 amino acids). In many other G-protein coupled receptors, ligand binding takes place within the 7TMD. However, the large ECD of each mGluR is thought to provide the ligand binding determinants (Nakanishi, Science 258:597, 1992; O""Hara et al., Neuron 11:41, 1993; Shigemoto et al., Neuron 12:1245, 1994). Chimeric mGluRs in which the ECDs of mGluRs with different ligand affinities and different G-protein coupling are exchanged have been used to demonstrate that the ECD of mGluRs defines ligand specificity but not G-protein specificity (Takahashi et al., J. Bio. Chem. 268:19341, 1993). Also unlike other G-protein coupled receptors in which the third intracellular loop is variable in size and sequence, the third intracellular loops of mGluRs are small and extremely well conserved (Brown E. M. et al., Nature 366:575, 1993). Chimeric mGluRs have been prepared in which the second intracellular loops and/or cytoplasmic tails were exchanged (Pin et al., EMBO J. 13:342). These experiments lead the investigators to conclude that unlike most other G-protein coupled receptors, xe2x80x9cboth the C-terminal end of the second intracellular loop and the segment located downstream of the seventh transmembrane domain are necessary for the specific activation of phospholipase-C by mGluR1cxe2x80x9d and to suggest that the second intracellular loop of mGluRs plays the role of the third intracellular loop of other G-protein coupled receptors.
Naturally occurring mRNA splice variants have been noted to produce prostaglandin E3 (EP3) receptors with essentially identical ligand binding properties but which preferentially activate different second messenger pathways (differential G-protein coupling) and which exhibit different desensitization properties (Namba et al., Nature 365:166, 1993; Sgimoto et al., J. Biol. Chem. 268:2712, 1993; Negishi et al., J. Biol. Chem. 268:9517, 1993). These variant receptor isoforms differ only in their cytoplasmic tails. The isoforms with the longer tails couple efficiently to phospholipase-C while those with the shorter tails do not. However, analyses of naturally occurring mRNA splice variants of mGluR1 and mGluR5 have indicated that their long cytoplasmic tails may not be directly involved in G protein coupling (Pin et al., Proc. Nat""l. Acad. Sci. U.S.A. 89:10331, 1992; Joly et al., J. Neuroscience 15:3970, 1995). In fact, Pin et al., (supra) have stated that xe2x80x9cThe very long C-terminal domain found only in PLC-coupled mGluRs (mGluR1 and 5) is, however, probably not involved in the specific interaction with PLC-activating G proteins.xe2x80x9d
Recently, calcium receptor has been described (Brown E. M. et al., Nature 366:575, 1993; Riccardi D., et al., Proc. Nat""l. Acad. Sci. USA 92:131-135, 1995; Garrett J. E., et al., J. Biol. Chem. 31:12919-12925, 1995). This CaR is the only known receptor which exhibits significant sequence homology with mGluRs except for other mGluRs. The CaR exhibits about xcx9c25% sequence homology (amino acid identities) to any one mGluR while mGluRs are  greater than 40% homologous (amino acid identities) to one another. The CaR is structurally related to mGluRs having a large ECD which has been implicated in receptor function and probable ligand binding (Brown E. M. et al., Nature 366:575, 1993; Pollak, M. R., et al., Cell 75:1297-1303, 1993). This similarity of structure does not confer close similarity in ligand binding specificity since the native ligand for the CaR is the inorganic ion, Ca2+, and glutamate does not modulate CaR activity. The CaR also has a large cytoplasmic tail and is coupled to the stimulation of phospholipase-C. Thus, the CaR is structurally and functionally more related to mGluR1 and 5 than to other mGluRs. Pin et al., (EMBO J. 13:342, 1994) have noted that certain amino acids are conserved within the intracellular loops of mGluRs which couple to phospholipase-C and different amino acids are conserved in these same positions within the intracellular loops of mGluRs which couple to the inhibition of adenylate cyclase. Intracellular loops 1 and 3 are the most highly conserved sequences between mGluRs and the CaR (Brown E. M. et al., Nature 366:575, 1993), but only about half of these particular amino acids are found in the corresponding position of the CaR and only one of these is actually the amino acid predicted for a receptor which couples to phospholipase-C. Thus, sequence conservation between CaRs and mGluRs appears to be consistent mostly with conservation of structural domains involved in ligand binding and G-protein coupling and does not provide evidence for specific sequence motifs within intracellular regions predictive of G-protein coupling specificity. Cell lines expressing CaRs have been obtained and their use to identify compounds which modulate the activity of CaRs disclosed.
An ideal screening procedure for identifying molecules specifically affecting the activity of different mGluRs would provide cell lines expressing each functional mGluR in such a manner that each was coupled to the same second messenger system and amenable to high throughput screening.
None of the references mentioned herein are admitted to be prior art to the claims.
The present invention concerns (1) chimeric receptor proteins having sequences from metabotropic glutamate receptors and calcium receptors, and fragments of metabotropic glutamate receptors, calcium receptors, and chimeric receptors; (2) nucleic acids encoding such chimeric receptor proteins and fragments; (3) uses of such receptor proteins, fragments and nucleic acids; (4) cell lines expressing such nucleic acids; (5) methods of screening for compounds that bind to or modulate the activity of metabotropic glutamate receptors or calcium receptors using such chimeric receptor proteins and fragments; (6) compounds for modulating metabotropic glutamate receptors or calcium receptors identified by such methods of screening; (7) methods for modulating metabotropic glutamate receptors or calcium receptors utilizing such compounds; and (8) methods of treating neurological disorders using such compounds.
A preferred use of the compounds and methods of the present invention is to screen for compounds which modulate metabotropic,glutamate receptor activity and to use such compounds to aid in the treatment of neurological diseases or disorders.
As described in the Background of the Invention above, metabotropic glutamate receptors and calcium receptors have similar structures. Both types of receptors have an extracellular domain (ECD), a seven transmembrane domain (7TMD) and an intracellular cytoplasmic tail (CT). Thus, in the chimeric receptors of the present invention, a portion of the sequence of the receptor is the same as or homologous to a portion of the sequence of an mGluR and a portion of the sequence is the same as or homologous to a portion of the sequence of a CaR. For example, the chimeric receptor can consist of the ECD of an mGluR and the 7TMD and CT of a CaR. Likewise, a chimeric receptor may include the ECD and 7TMD of an mGluR and the CT of a CaR. Other combinations of mGluR and CaR domains or portions of domains may also be constructed and utilized. These chimeric receptors are of interest, in part, because they allow the coupling of certain functional aspects of an mGluR with certain functional aspects of a CaR. Thus, experiments have shown that ligands known in the art which are agonists or antagonists on a native mGluR also exhibit such activities on chimeric receptors in which the extracellular domain is from the mGluR. Similarly, experiments have shown that ligands known in the art which modulate mGluRs act on chimeric receptors in which the extracellular domain and the 7TMD are from an mGluR. In both of these cases, it is expected that other ligands which modulate mGluR activity will also act on these types of chimeric receptors.
The use of mGluRs for screening for mGluR active compounds has been complicated by a number of factors including a rapid desensitization of the receptor upon ligand binding/activation and difficulties in stably expressing the receptors in recombinant vertebrate cells (see, for example, FIG. 8B and also published PCT Patent Application). Certain of the chimeric receptors of the present invention can be utilized to overcome these technical difficulties and provide much improved screening methods by utilizing the more robust aspects of calcium receptors. For example, by coupling the 7TMD and the CT of the CaR with the extracellular domain of an mGluR, or the CT of the CaR to the ECD and 7TMD of the mGluR, the mGluR extracellular domain has the benefit of the Gq coupling property of a CaR as well as the improved property of a lack of rapid densensitization (see, for example, FIG. 8C). Thus, such a chimeric receptor has the ligand binding and activation properties similar to those of a native mGluR but having the improved second messenger coupling similar to a CaR. Therefore, the chimeric receptor simplifies and enables efficient, practical, and reproducible functional screens to identify mGluR active molecules.
For these novel chimeric receptors, not only is the combination of mGluR and CaR sequences in a chimeric receptor novel, but also the successful coupling of the activities is unexpected. Previously, such coupling had only been accomplished using portions of receptors with closely related sequences. In this case the sequence identity between metabotropic glutamate receptors and calcium receptors is only about 19-25%, and the two types of receptors share only a 25-30% sequence similarity (Brown et al., Nature 366:575, 1993).
It is recognized that the three domains described above are made up of sub-domains, for example, ligand binding sites and G protein coupling sites. Therefore, for some applications it is not necessary to include in a chimeric receptor a complete domain from a particular receptor in order to have the desired activity. For example, only the ligand binding site from an mGluR can be incorporated in a chimeric receptor in which most or all of the remainder of the sequence is homologous to a CaR. Likewise, in a chimeric receptor, one of the cytoplasmic loops of the 7TMD can be homologous to a loop sequence of an mGluR and substantially the remainder of the sequence of the receptor can be homologous to a CaR, or conversely, one of the cytoplasmic loops can be homologous to a loop sequence of a CaR and substantially the remainder of the sequence of the receptor can be homologous to an mGluR.
Thus, in a first aspect the invention features a composition including a chimeric receptor which has an extracellular domain, a seven transmembrane domain, and an intracellular cytoplasmic tail domain. The chimeric receptor has a sequence of at least 6 contiguous amino which is homologous to a sequence of a metabotropic glutamate receptor and a sequence of at least 6 contiguous amino acids which is homologous to a sequence of a calcium receptor.
In preferred embodiments, at least one domain is homologous to a domain of a metabotropic glutamate receptor, or at least one domain is homologous to a domain of a calcium receptor. In particular, this includes chimeric receptors having a domain homologous to a metabotropic glutamate receptor and a domain homologous to a calcium receptor.
Also in preferred embodiments, the chimeric receptor has two domains from a metabotropic glutamate receptor and one domain from a calcium receptor, or two domains from a calcium receptor and one domain from a metabotropic glutamate receptor. This includes each of the possible combinations of the three domains. For example, in a more preferred embodiment, the chimeric receptor has one domain homologous to the extracellular domain of a metabotropic glutamate receptor, one domain homologous to the seven transmembrane domain of a metabotropic glutamate receptor, and one domain homologous to the intracellular cytoplasmic tail domain of a calcium receptor.
In other preferred embodiments, the chimeric receptor has at least one cytoplasmic loop of the seven transmembrane domain which is homologous to a cytoplasmic loop of a metabotropic glutamate receptor. Similarly, in other preferred embodiments, the chimeric receptor has at least one cytoplasmic loop homologous to a cytoplasmic loop of a calcium receptor.
Also in other preferred embodiments, the chimeric receptor has a sequence of at least 6 contiguous amino acids which is homologous to an amino acid sequence of a calcium receptor, and the rest of the sequence of the chimeric receptor is homologous to an amino acid sequence of a metabotropic glutamate receptor. In other embodiments, the sequence homologous to an amino acid sequence of a calcium receptor may beneficially be longer, for example at least 12, 18, 24, 30, 36, or more amino acids in length.
Similarly, in other preferred embodiments, the chimeric receptor has a sequence of at least 6 contiguous amino acids which is homologous to an amino acid sequence of a metabotropic glutamate receptor, and the rest of the sequence of the chimeric receptor is homologous to an amino acid sequence of a calcium receptor. In other embodiments, the sequence homologous to an amino acid sequence of a metabotropic glutamate receptor may beneficially be longer, for example at least 12, 18, 24, 30, 36, or more amino acids in length.
In a related aspect, the invention provides a composition which includes an isolated, enriched, or purified nucleic acid molecule which codes for a chimeric receptor as described for the aspect above. In particular, this includes nucleic acid coding for a chimeric receptor having a sequence of at least 6 contiguous amino acids which is homologous to an amino acid sequence of a calcium receptor and a sequence of at least 6 contiguous amino acids which is homologous to an amino acid sequence of a metabotropic glutamate receptor. Similarly to the above aspect, in particular embodiments the chimeric receptor sequence homologous to an amino acid sequence from a calcium receptor and/or a metabotropic glutamate receptor may beneficially be longer, for example, at least 12, 18, 24, 30, 36, or more amino acids in length.
In preferred embodiments, the chimeric receptor has a domain homologous to a domain of a metabotropic glutamate receptor, and/or a domain homologous to a calcium receptor. In more preferred embodiments, the chimeric receptor has two domains homologous to domains of a metabotropic glutamate receptor and a domain homologous to a domain of a calcium receptor, or two domains homologous to domains of a calcium receptor and a domain homologous to a domain of a metabotropic glutamate receptor.
In another related aspect, the nucleic acid encoding a chimeric receptor, as described above, is present in a replicable expression vector. Thus, the vector can include nucleic acid sequences coding for any of the chimeric receptors described.
Also in a related aspect, the invention provides a recombinant host cell transformed with a replicable expression vector as described above.
The invention also features a process for the production of a chimeric receptor; the process involves growing, under suitable nutrient conditions, procaryotic or eucaryotic host cells transformed or transfected with a replicable expression vector containing a nucleic acid sequence coding for a chimeric receptor as described above, in a manner allowing expression of the chimeric receptor.
By xe2x80x9cisolatedxe2x80x9d in reference to a nucleic acid is meant the nucleic acid is present in a form (i.e., its association with other molecules) other than found in nature. For example, isolated receptor nucleic acid is separated from one or more nucleic acids which are present on the same chromosome. Preferably, the isolated nucleic acid is separated from at least 90% of the other nucleic acids present on the same chromosome. Preferably, the nucleic acid is provided as a substantially purified preparation representing at least 75%, more preferably 85%, most preferably 95% of the total nucleic acids present in the preparation.
Another example of an isolated nucleic acid is recombinant nucleic acid. Preferably, recombinant nucleic acid contains nucleic acid encoding a chimeric metabotropic glutamate receptor or metabotropic glutamate receptor fragment cloned in an expression vector. An expression vector contains the necessary elements for expressing a cloned nucleic acid sequence to produce a polypeptide. An expression vector contains a promoter region (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal synthesis initiation. xe2x80x9cExpression vectorxe2x80x9d includes vectors which are capable of expressing DNA sequences contained therein, i.e., the coding sequences are operably linked to other sequences capable of effecting their expression. It is implied, although not always explicitly stated, that these expression vectors must be replicable in the host organisms either as episomes or as an integral part of the chromosomal DNA.
A useful, but not a necessary, element of an effective expression vector is a marker encoding sequencexe2x80x94i.e., a sequence encoding a protein which results in a phenotypic property (e.g. tetracycline resistance) of the cells containing the protein which permits those cells to be readily identified. In sum, xe2x80x9cexpression vectorxe2x80x9d is given a functional definition, and any DNA sequence which is capable of effecting expression of a specified contained DNA code is included in this term, as it is applied to the specified sequence. As at present, such vectors are frequently in the form of plasmids, thus xe2x80x9cplasmidxe2x80x9d and xe2x80x9cexpression vectorxe2x80x9d are often used interchangeably. However, the invention is intended to include such other forms of expression vectors, including viral vectors, which serve equivalent functions and which may, from time to time become known in the art. Recombinant nucleic acids may contain nucleic acids encoding for a chimeric metabotropic glutamate receptor, receptor fragment, or chimeric metabotropic glutamate receptor derivative, under the control of its genomic regulatory elements, or under the control of exogenous regulatory elements including an exogenous promoter. By xe2x80x9cexogenousxe2x80x9d is meant a promoter that is not normally coupled in vivo transcriptionally to the coding sequence for the metabotropic glutamate receptor or calcium receptor.
The invention also provides methods of screening for compounds which bind to and/or modulate the activity of a metabotropic glutamate receptor and/or a calcium receptor. These methods utilize chimeric receptors as described above or nucleic acid sequence encoding such chimeric receptors. Such chimeric receptors provide useful combinations of characteristics from the two types of receptors, such as combining the binding characteristics from a metabotropic glutamate receptor with the cellular signaling characteristics from a calcium receptor.
Thus, in another aspect the invention provides a method of screening for a compound that binds to or modulates the activity of a metabotropic glutamate receptor. The method involves preparing a chimeric receptor having an extracellular domain, a seven transmembrane domain, and an intracellular cytoplasmic tail domain, in which at least one domain is homologous to a domain of a metabotropic glutamate receptor and at least one domain is homologous to a domain of a calcium receptor. The chimeric receptor and a test compound are introduced into an acceptable medium. The binding of a test compound to the chimeric receptor, or the modulation of the chimeric receptor by the compound, is monitored by physically detectable means to identify those compounds which bind to or modulate the activity of a metabotropic glutamate receptor.
In a preferred embodiment the extracellular domain of the chimeric receptor is homologous to the extracellular domain of a metabotropic glutamate receptor. Also in preferred embodiments, the chimeric receptor has two domains homologous to domains of a metabotropic glutamate receptor and a domain homologous to a domain of a calcium receptor, or two domains homologous to domains of a calcium receptor and a domain homologous to a domain of a metabotropic glutamate receptor.
In another aspect the invention provides a method of screening for a compound which binds to or modulates the activity of a metabotropic glutamate receptor, utilizing a nucleic acid coding for a chimeric receptor. This method involves preparing a nucleic acid sequence encoding a chimeric receptor which has an extracellular domain, a seven transmembrane domain and an intracellular cytoplasmic tail domain, in which the chimeric receptor has a sequence of at least six contiguous amino acids which is homologous to a sequence of amino acids of a calcium receptor and a sequence of at least six contiguous amino acids which is homologous to a sequence of amino acids of a metabotropic glutamate receptor. The nucleic acid sequence is inserted into a replicable expression vector capable of expressing the chimeric receptor in a host cell. A suitable host cell is transformed with this vector and the transformed host cell and a test compound are introduced into an acceptable medium. Identification of binding or modulation by the test compound is performed by monitoring the effect of the compound on the cell.
In a preferred embodiment the chimeric receptor has at least one domain homologous to a domain of metabotropic glutamate receptor and/or at least one domain homologous to a domain of a calcium receptor. In particular this includes preferred embodiments in which the chimeric receptor has an extracellular domain homologous to an extracellular domain of a metabotropic glutamate receptor and/or a seven transmembrane domain of a metabotropic glutamate receptor. In particular embodiments, the chimeric receptor has two domains homologous to domains of a metabotropic glutamate receptor and a domain homologous to a domain of a calcium receptor, or two domains homologous to domains of a calcium receptor and a domain homologous to a domain of a metabotropic glutamate receptor.
Also in a preferred embodiment the chimeric receptor has at least one cytoplasmic loop of the seven transmembrane domain which is homologous to a cytoplasmic loop of a calcium receptor; in particular embodiments the sequence of the remainder of the chimeric receptor is homologous to the sequence of a metabotropic glutamate receptor.
In another preferred embodiment the chimeric receptor has a sequence of at least six contiguous amino acids which is homologous to a sequence of amino acids of a calcium receptor and the remainder of the amino acids sequence of a chimeric receptor is homologous to an amino acid sequence of a metabotropic glutamate receptor. In yet another preferred embodiment the chimeric receptor has at least one cytoplasmic loop of the seven transmembrane domain which is homologous to a cytoplasmic loop of a metabotropic glutamate receptor.
In still another preferred embodiment the host cell is a eucaryotic cell.
In the context of the methods of this invention, xe2x80x9cmonitoring the effectxe2x80x9d of a compound on a host cell refers to determining the effects of the compound on one or more cellular processes or on the level of activity of one or more cellular components, or by detection of an interaction between the compound and a cellular component.
The invention also provides methods of screening for compounds that bind to or modulate a metabotropic glutamate receptor or calcium receptor using fragments of such receptors. Such fragments can, for example, be chosen to include a sequence which has been shown to be important in activation of the receptor""s signal pathway.
Thus, in another aspect the invention features a method of screening for a compound that binds to a metabotropic glutamate receptor or a calcium receptor, by preparing a nucleic acid encoding a fragment of such a receptor, inserting the sequence into a replicable expression vector which can express that fragment in a host cell, transforming a suitable host cell with a vector, recovering the fragment from the host cell, introducing the fragment in a test compound into an acceptable medium and monitoring the binding of the compound to the fragment by physically detectable means.
In a preferred embodiment the fragment is a fragment of a metabotropic glutamate receptor; in a more preferred embodiment the fragment includes the extracellular domain of that receptor.
In another preferred embodiment the fragment includes the seven transmembrane domain of a metabotropic glutamate receptor. In a more preferred embodiment the fragment includes both the seven transmembrane domain and the cytoplasmic tail domain of a metabotropic glutamate receptor.
Similarly in another preferred embodiment the fragment is a fragment of a calcium receptor, preferably including the extracellular domain over the seven transmembrane domain of that receptor. In a more preferred embodiment the fragment includes the seven transmembrane domain and cytoplasmic tail domain of the calcium receptor.
Certain receptor fragments are able to activate one or more cellular responses in a manner similar to the receptor from which the fragment was derived. Therefore, in a related aspect, the invention provides a method of screening for a compound that binds to or modulates a metabotropic glutamate receptor or a calcium receptor by preparing a nucleic acid sequence encoding a fragment of such a receptor, inserting that sequence into a replicable expression vector, transforming a host cell with that vector, introducing the host cell and a test compound into an acceptable medium, and monitoring the effect of the compound on the host cell.
For certain receptors it is possible to utilize fragments of two different receptors to screen for compounds which bind to or modulate a receptor. The method involves preparing a nucleic acid encoding a fragment of a first receptor, inserting the sequence into a replicable expression vector capable of expressing that fragment in a host cell, transforming a suitable host cell with a vector, and recovering the first fragment from the host cell. A fragment of a second receptor is prepared in a similar manner. The two fragments and a test compound are introduced into an acceptable medium and the binding and/or modulation by the compound is monitored by physically detectable means.
In preferred embodiments a fragment is from a metabotropic glutamate receptor and a fragment is from a calcium receptor. In particular preferred embodiments the first fragment includes the extracellular domain of a metabotropic glutamate receptor and the second fragment includes the seven transmembrane domain and cytoplasmic tail domain of a calcium receptor, or the first fragment includes the extracellular domain and the seven transmembrane domain of a metabotropic glutamate receptor and the second fragment includes the cytoplasmic tail domain of a calcium receptor.
In another particular embodiment the first fragment includes the extracellular domain of a calcium receptor and the second fragment includes the seven transmembrane domain and the cytoplasmic tail domain of a metabotropic glutamate receptor. In still another particular preferred embodiment, the first fragment includes the extracellular domain of a calcium receptor and the second fragment includes the seven transmembrane domain of a metabotropic glutamate receptor and the cytoplasmic tail domain of a calcium receptor.
Certain compounds can be identified which modulate the activity of both a metabotropic glutamate receptor and of a calcium receptor. Thus, this invention also provides a method for screening for such compounds by preparing a nucleic acid sequence encoding a chimeric receptor which includes a domain homologous to a domain of a metabotropic glutamate receptor and a domain homologous to a domain of a calcium receptor. The sequence is inserted in a replicable expression vector capable of expressing the receptor in a host cell; a suitable host cell is transformed with the vector and the transformed host cell and a test compound are introduced into an acceptable medium. The binding or modulation by the compound is observed by monitoring the effect of a compound on the host cell.
The invention also provides methods for determining the site of action of a compound active on a metabotropic glutamate receptor or on a calcium receptor. The methods involve preparing a nucleic acid sequence which encodes a chimeric receptor. In two related aspects, a chimeric receptor has at least a six amino acid sequence which is homologous to a sequence of amino acids of a calcium receptor and the remainder of the amino acid sequence is homologous to an amino acid sequence of a metabotropic glutamate receptor, or the chimeric receptor has at least a six amino acid sequence which is homologous to a sequence of amino acids of a metabotropic glutamate receptor and the remainder of amino acid sequence is homologous to a sequence of a calcium receptor. In these aspects, the nucleic acid sequence is inserted into a replicable expression vector which is capable of expressing the receptor in a host cell. The vector is transformed into a suitable host cell and the transformed host cell in the compound are introduced into an acceptable medium. The effect of the compound on the host cell is monitored; thus if a compound is active on a receptor through an interaction at the sequence of at least six amino acids from the corresponding receptor, the chimeric receptor will be activated and the cellular effects can be observed. On the other hand if the compound does not interact with the at least six amino acid sequence, thereby activating the receptor, the corresponding cellular effects will not be observed.
Thus, xe2x80x9csite of actionxe2x80x9d refers to the location(s) on the receptor which are involved in interaction with a natural ligand for that receptor, or with another compound of interest. For example, for a compound which modulates the activity of a metabotropic glutamate receptor by binding to the receptor, the site of action can include amino acid sequences associated with binding of the compound to the receptor, but may also include other sequences. Such other sequences can, for example, include sequences whose secondary or tertiary structure is altered in response to the binding of the compound.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments, and from the claims.